We propose to develop a molecular imaging probe that will provide quantitative information on the[unreadable] expression level of mRNA with spatial and temporal resolution. Specifically, an oligonucleotide-based probe[unreadable] will be designed to form a stem-loop structure and will be labeled with a 'reporter' fluorophore at one end and[unreadable] a quencher at the other, analogous to a molecular beacon; however, the oligonucleotide will also be labeled[unreadable] with a second optically distinct 'reference' dye/nanoparticle, which will be selected such that it is unquenched[unreadable] regardless of the conformation of the probe. Fluorescently labeled neutravidin and quantum dots will be[unreadable] tested for their suitability in serving as the reference dye. We hypothesize that beneficial features of this[unreadable] novel probe compared with conventional molecular beacons will include (1) the ability to monitor transfection[unreadable] efficiency due to the presence of the unquenched reference dye. This will reduce false-negatives by[unreadable] allowing for the differentiation between untransfected cells and cells with low levels of gene expression. (2)[unreadable] The ability to remove via ratiometric imaging (i.e. reporter fluorscence/reference fluorescence) the impact of[unreadable] instrumental and experimental variability. (3) The ability to quantitatively compare variations in gene[unreadable] expression levels between samples, between cells within individual samples, and even between sub-cellular[unreadable] compartments by using the reference dye as a point of reference (4) The ability to quantify gene expression[unreadable] with spatial and temporal resolution since the covalent linkage between the reporter and reference dye[unreadable] ensures they exhibit an equivalent intracellular lifetime and co-localization pattern. (5) The ability to use the[unreadable] quantum dot/neutravidin as a platform to attach targeting agents, opening up the possibility for in vivo[unreadable] imaging. (6) The possibility of an improved signal-to-background due to quenching of the 'reporter' dye by[unreadable] both the quencher molecule and the 'reference' dye. To evaluate these features we will pursue two major[unreadable] aims during the proposed research: 1) We will design, synthesize and characterize the 'quantitative'[unreadable] molecular beacon (QMB) in terms of its signal-to-background and lower detection limit (in vitro and in vivo)[unreadable] and 2) we will evaluate the ability of the QMBs to quantify endogenous mRNA expression in breast cancer[unreadable] cells in real-time. It is envisioned that the approach proposed here will allow significant advancements in our[unreadable] understanding of human health and disease and could potentially prove to be a powerful diagnostic tool.